Functional characterization of two S-nitroso-l-cysteine transporters, which mediate movement of NO equivalents into vascular cells

Abstract

Li S, Whorton AR. Functional characterization of two S-nitroso-Lcysteine transporters, which mediate movement of NO equivalents into vascular cells. Am J Physiol Cell Physiol 292: C1263-C1271, 2007. First published November 8, 2006; doi:10.1152/ajpcell.00382.2006.-System L amino acid transporters have been shown to be responsible for cellular uptake of S-nitroso-L-cysteine (L-CSNO). In this study, we examined the characteristics of L-CSNO uptake in Xenopus laevis oocytes expressing system L transporters and found that uptake increased only when both 4F2 heavy chain (4F2HC) and either L-type amino acid transporter 1 (LAT1) or LAT2 light chain were coexpressed. The K m for transport was 57 Ϯ 8 M for 4F2HC-LAT1 and 520 Ϯ 52 M for 4F2HC-LAT2. Vascular endothelial and smooth muscle cells were shown to express transcripts for 4F2HC and for both LAT1 and LAT2. Transport of L-CSNO into red blood cells, endothelial cells, and smooth muscle cells was inhibited by 2-aminobicyclo(2.2.1)heptane-2-carboxylic acid (BCH) and by large neutral amino acids demonstrating functional system L transporters in each cell type. Uptake of L-CSNO led to accumulation of cellular S-nitrosothiols and inhibition of both growth factor-induced ERK phosphorylation and TNF-␣-mediated IB degradation. Similar effects were seen when cells were incubated simultaneously with S-nitrosoalbumin and L-cysteine but not with D-cysteine or with S-nitrosoalbumin alone. In each case, nitrosylation of proteins and cellular responses were blocked by BCH. Together, these data suggest that transmembrane movement of nitric oxide (NO) equivalents from the plasma albumin NO reservoir is mediated by cysteine, which serves as a carrier. The mechanism requires transnitrosylation from S-nitrosoalbumin to free cysteine and activity of system L transporters, thereby providing a unique pathway for cellular responses to S-nitrosoalbumin. nitric oxide; nitrosothiols; system L transporter; endothelial cells; smooth muscle cells; red blood cells NITRIC OXIDE (NO) has been implicated in numerous cellular functions, from vascular smooth muscle relaxation to regulation of gene expression (11,19,26). The short half-life of NO can be prolonged by reaction with sulfhydryl groups of proteins or low molecular thiols to form more stable, biologically active S-nitrosothiols (23,32). Although the actual concentration in the circulation is debated, S-nitrosothiols are known to be formed in vivo (8,10,17,31,33,39) and are proposed to mediate diverse biological responses (7, 9). Among the circulating S-nitrosothiols, S-nitrosoalbumin is an important species and may act as reservoir of NO (8,31,34,35). The physiological roles for S-nitrosoalbumin or mechanisms for transfer of NO equivalents from this circulating S-nitrosoprotein to vascular tissues are not known. However, mobilization of NO bioactivity from S-nitrosoalbumin by low-molecular-weight thiols leads to vasorelaxation (13, 29), and low-molecularweight thiols are involved in the ability of S-nitrosoalbumin to inhibit platelet function...

Nitric Oxide Transport in Blood: A Third Gas in the Respiratory Cycle

“…An O 2 -linked change in Hb conformation also initiates export of SNO from RBCs by promoting NO transfer to receptor thiols including those associated with the erythrocytic membrane protein AE-1 (band 3) (226) and thence to extra-erythrocytic thiols (64,223) to form plasma or other cellular SNOs, which are vasoactive at concentrations as low as 1 to 5 nM (149,262). In further support of this paradigm, two SNO-cysteine transporters, which may mediate the movement of NO equivalents (as vasoactive low-mass SNOs) into endothelial cells (175), have been recently discovered.…”

“…Moreover, perhaps to facilitate compensatory transmembrane SNO traffic (174,175,284,314), increased capacity for cationic amino acid transport (system y+ and y + L) has been noted in RBCs from humans with chronic heart failure (112,200). Increased capacity for peripheral SNO flux would promote augmented NO delivery by RBCs to the periphery in response to global perfusion insufficiency (e.g., result in generalized afterload reduction).…”

S-Nitrosylation of Sarcomeric Proteins Depresses Myofilament Ca2+ Sensitivity in Intact Cardiomyocytes

“…Besides the shorter exposure time in this experiment, there was an intact sarcolemma; CysNO can only cross this barrier by active transport (25). Half-maximal intracellular effects achieved when CysNO is added to the medium require between 20 and 180 lM for different cells from the aorta (8,33). Thus, the lower sensitivity in our immunoblots for MLC3 and Tm may reflect a kinetic limitation; CysNO had only 10 min to cross the cell membrane and react with thiols rather than the 60 min allotted for GSNO in demembranated myofibrils.…”

S‐nitrosothiols as selective antithrombotic agents – possible mechanisms

“…Evidence has emerged from a number of different laboratories showing that the low molecular weight RSNO compounds cysNO and S-nitrosohomocysteine act as substrates for the widely-distributed amino acid transporter system-L (L-AT). This mechanism for transmembrane transport of cysNO could explain why stereoselective haemodynamic effects are seen following administration of L-cysNO and D-cysNO to rats (Davisson et al, 1996), and experimental studies carried out in vitro have confirmed its presence in a number of cell types including erythrocytes (Sandmann et al, 2005), endothelial cells (Broniowska et al, 2006), vascular smooth muscle cells (Li and Whorton, 2007;Riego et al, 2009), epithelial cells (Granillo et al, 2008), and various transformed cell lines (Zhang and Hogg, 2004;Li and Whorton, 2005), although to date, there has been no direct demonstration of cysNO uptake via L-AT in platelets. In these published studies, other forms of RSNOs, including GSNO, S-nitrosocysteinyl-glycine, S-nitroso-N-acetyl-penicillamine and S-nitrosoalbumin, failed to be transported via L-AT, nor could they mediate NO-related signalling in target cells unless extracellular cysteine was supplied.…”